JPH0583081B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to a method for purifying and separating branched cyclodextrin from a solution containing branched cyclodextrin, and more specifically to a method for purifying branched β-cyclodextrin. and regarding separation. BACKGROUND OF THE INVENTION Starch is a polymer of anhydroglucose;
It is present in various plants such as corn, potatoes, sorghum, and rice. Regardless of the plant variety, starch is extracted chemically as starch amylose (linear) and amylopectin (branched), which exist in two forms. Amylose is a linear polymer of anhydroglucose units linked together by α-1,4 bonds, whereas amylopectin is a linear polymer of α-1,4 anhydroglucose on a linear polymer of α-1,4 anhydroglucose. It consists of glucose polymer side chains attached. In amylopectin, the bond between the linear chain and the side chain is an α-1,6 bond. Starches generally contain both amylose and amylopectin molecules, the amounts of which depend on the origin of each starch. For example, high amylose corn starch has an amylopectin to amylose ratio of about 50:50, while waxy maize corn starch has an amylopectin to amylose ratio of 99:1. Generally, most starches contain some amylopectin. Cyclodextrins, also referred to as Schaldinger's dextrin, cycloamylose, cyclomaltose, and cycloglucan, are polymers of anhydroglucose that are linked together by alpha-1,4 bonds to form cyclic compounds. The 6-membered ring is referred to as α-cyclodextrin, the 7-membered ring as β-cyclodextrin, and the 8-membered ring as γ-cyclodextrin. Also, these 6,7
and the 8-membered ring are also referred to as cyclomaltohexaose, cyclomaltoheptaose and cyclomaltooctaose, respectively. Branched cyclodextrins were described by French and his collaborators as early as 1965 (French et al., Archives of Biochem.
Biophys. Vol. 3, pp. 153-160, 1965.)
However, there has been little research recently. Branched β-cyclodextrins, as these names imply, contain at least one unit of anhydroglucose on an unbranched β-cyclodextrin.
It is a bond that extends outward from the branched ring structure. The branches are linked back by alpha-1,6 linkages, the same linkages that amylopectin uses to link side chains onto its linear chains. By definition, "β-" without the modifier "branched" or "unbranched"
The term "cyclodextrin" refers to both branched and unbranched β-cyclodextrins. For branched and unbranched β-cyclodextrins,
I will call them as they are. Cyclodextrins have a wide variety of uses as they can act as hosts for a large number of compounds and have already found use in the agricultural and pharmaceutical fields as well as other fields. Branched β-cyclodextrin is more soluble in water than unbranched β-cyclodextrin. Specifically, unbranched β-cyclodextrin has a solubility of about 2% by weight, whereas branched β-cyclodextrin has a
It has a solubility of about 50% and more by weight.
By using branched β-cyclodextrin as a host molecule for insoluble compounds,
Insoluble compounds can be dissolved in water. Another important aspect of branched β-cyclodextrins is that the branches themselves can be used for other specialized applications of β-cyclodextrin.
The purpose is to provide a site for forming cyclodextrin derivatives. Generally, addition onto a branch does not interfere with the ring structure of the β-cyclodextrin, allowing the ring structure of the branched β-cyclodextrin to function as a host molecule. Generally, branching per se does not interfere with the function of the ring as a host molecule. A solution containing β-cyclodextrins is
It is produced by treating a starch slurry with the enzyme cyclodextrin glycosyltransferase (CGT) at a pH, temperature and time appropriate for the CGT chosen. Starch is
It can be derived from any selected plant species. The enzyme CGT can be used in microorganisms such as Bacillus macerans, B. megaterium, B. circulans, B. stearothermophilus (B.
stearothermophilus) and Bacillus sp. (alkaliphilic) and others. The parameters between the CGT enzyme chosen and the starch chosen are conventional and well described in the literature. Generally, starch is slurried in an aqueous solution at a concentration of up to about 35% by weight solids. Next, the slurry is
Subjected to gelation and liquefaction by enzymes or acids to about 1-5 DE. A preferred enzyme for liquefaction is bacterial alpha-amylase. Next, the CGT of choice is added to the gelling and liquefying slurry, and the PH, temperature and treatment time are adjusted depending on the enzyme of choice. Generally, the PH is 4.5 to approx.
8.5, the temperature range is between room temperature and about 75°C, and the reaction length is about 10 hours to
It will be held for 7 days. The amount of β-cyclodextrins will vary depending on the processing conditions and enzymes used. The unbranched β-cyclodextrin is then removed from the reaction solution by conventional crystallization or filtration. Recently, it has been believed that branched β-cyclodextrins are formed by the action of CGT on amylopectin in a conventional manner. Primarily to produce β-cyclodextrin, the reaction between CGT and gelled and liquefied starch slurry is carried out in the presence of a solvent such as toluene or p-xylene. Such solvents substantially increase the yield of β-cyclodextrin. Generally, when starch is treated with CGT, branched β-cyclodextrin, unbranched β-
Cyclodextrins and acyclic dextrins are produced. The separation and purification of branched β-cyclodextrins from solution is extremely difficult, and the methods used traditionally are very complex and It has not been possible to separate branched β-cyclodextrins from formula dextrins on a large scale. Needless to say, the only known methods for separating and purifying branched β-cyclodextrins from unbranched and acyclic dextrins are special chromatographic methods; is impractical for truly industrial operation. SUMMARY OF THE INVENTION A practical method for separating and purifying branched β-cyclodextrin from unbranched β-cyclodextrin and acyclic dextrin has now been invented. Broadly, the purification and separation methods of the present invention:
A primary precipitate and a primary mother liquor are formed from the starting aqueous solution as a first solution by adding a β-cyclodextrin clathrate-forming complex to the starting aqueous solution containing the branched β-cyclodextrin. collecting a primary precipitate from the first solution; forming a second aqueous solution with the primary precipitation; secondary precipitation from the second solution by adding a compound forming a β-cyclodextrin clathrate to the second solution; and forming a secondary mother liquor; recovering the secondary mother liquor; and finally recovering branched β-cyclodextrin from said secondary mother liquor. Compounds that form β-cyclodextrin clathrates are well known chemicals. Preferably, the primary precipitate is washed before forming the second solution. Such a washing step involves washing the precipitate using a small amount of water and removes at least some acyclic dextrin from the primary precipitate. Also preferably, in order to increase the purity of the branched β-cyclodextrin, instead of recovering the branched β-cyclodextrin from the secondary mother liquor, a third solution is formed from the secondary mother liquor, and - adding a compound forming a cyclodextrin clathrate to said third solution to form a tertiary precipitate and a tertiary mother liquor, recovering the tertiary mother liquor, and finally preparing a branched β-cyclodextrin from said tertiary mother liquor; to recover. The starting aqueous solution or first solution used in the present invention is about 1% or more of the total solids content of the solution is branched β-cyclodextrin;
and unbranched β-cyclodextrin is an aqueous solution containing branched β-cyclodextrin and unbranched β-cyclodextrin such that an excess amount of branched β-cyclodextrin is present. The starting aqueous solution or first solution is a starch slurry that is processed using a CGT enzyme to produce a specific
It can be formed in a conventional manner by treating at a pH, temperature and length of time appropriate for the CGT enzyme. More preferably, the starting or first solution is treated with a CGT enzyme at a pH, temperature and length of time appropriate for the particular CGT enzyme and then subjected to conventional crystallization or filtration procedures to obtain unbranched β-
Obtained from products that have been treated to remove cyclodextrin. More specifically, for starch slurry
The action of CGT makes it possible to use the mother liquor remaining after industrial production of β-cyclodextrin.
A very good starting point or first step for the method of the invention
It was found that it becomes a solution. Preferably, the starch used in forming the first or starting solution of the present invention is waxy starch, and more preferably waxymaize. To obtain predominantly branched β-cyclodextrin, treatment of starch slurry with CGT is necessary to obtain
For example, it is carried out in the presence of toluene or p-xylene. Specifically, a preferred starting or first solution according to the invention comprises from about 1% to about 2% by weight of branched β-cyclodextrin and unbranched β-cyclodextrin.
Contains about 5% to about 20% cyclodextrin by weight. A compound forming a β-cyclodextrin clathrate is added to this starting aqueous solution to form a primary precipitate and a primary mother liquor. Compounds that form β-cyclodextrin clathrates combine with β-cyclodextrin to form clathrates that precipitate out of solution. This precipitate of clathrate compounds is called a primary precipitate. Addition of the compound forming the β-cyclodextrin clathrate is accomplished in a conventional manner using conventional equipment. Specifically, the compound forming the β-cyclodextrin clathrate is added to the solution while stirring the solution. The temperature of the stirred solution is preferably maintained between about 10 and about 25°C, more preferably between about 15 and about 20°C. The first or starting solution to which the compound forming the β-cyclodextrin clathrate is added is
It is left standing for about 1 day to about 7 days to form a precipitate. Those skilled in the art will understand that precipitation does not occur instantaneously, but after a period of time. It was found that all or most of the primary precipitate had formed after about 3 days. Most preferably, the period for precipitate formation is about 3 days or more. The concentration of the starting aqueous solution is preferably about 24% to about 40% by weight solids, more preferably about 40% by weight solids.
It is 30%. The amount of clathrate-forming compound added in the primary addition of β-cyclodextrin clathrate-forming compound, based on 100 parts by weight of β-cyclodextrin in solution, is at least about
10 parts by weight, and preferably β-
Approximately 20 parts based on 100 parts by weight of cyclodextrin
Parts by weight. The primary precipitate contains both branched and unbranched β-cyclodextrin. Compounds that form β-cyclodextrin clathrates are well known to those skilled in the art. Preferably p-xylene or toluene is used to add to the first or starting solution, most preferably p-
Xylene is used. Recovery of this primary precipitate is accomplished in a conventional manner using conventional equipment. Filters or centrifuges can be used, with filters being preferred. This step produces the primary mother liquor as a by-product. Formation of the second solution is accomplished by mixing water and the primary precipitate in a conventional manner using conventional equipment. Preferably, the precipitate is redissolved in water by heating and stirring the precipitate, and by solids weight approximately
Form a 1.0% to about 20%, more preferably about 10% by weight solution. The amount of heat required to completely dissolve the primary precipitate is such that the second solution is boiled and some of the β-cyclodextrin clathrate-forming compounds contained in the primary precipitate evaporate. It was discovered that it is something. Preferably, the precipitate is washed in a conventional manner to remove some of the acyclic dextrin before redissolving the precipitate. Washing of the primary precipitate is accomplished using 1 or 2 ml of water per gram of precipitate. Washing is 2
It is repeated three times. Secondary precipitation and formation of a secondary mother liquor is accomplished by adding to the second solution a compound that forms a β-cyclodextrin clathrate. β to the second solution
- The addition of the compound forming the cyclodextrin clathrate is carried out analogously to the addition of the compound forming the β-cyclodextrin clathrate to the first or starting solution. Based on 100 parts by weight of β-cyclodextrin in solution, at least about 10 parts by weight, and preferably about 20 parts by weight, of the compound forming the β-cyclodextrin clathrate are added. The preferred temperature of the second solution throughout the formation of the secondary precipitate is between about 10<0>C and about 25<0>C, more preferably between about 15<0>C and about 20<0>C. The period of secondary precipitate formation is preferably about 3 days or more. A preferred β-cyclodextrin clathrate-forming compound used in the secondary precipitation and secondary mother liquor formation is toluene. Recovery of the secondary mother liquor from the second addition of compounds forming the β-cyclodextrin clathrate is accomplished in a conventional manner using conventional equipment, such as centrifuges or filters. Preferably, the secondary precipitate is filtered off,
Leave only the filtrate. This filtrate or mother liquor contains branched and some unbranched β-cyclodextrins that are not clathrates and form secondary precipitates. The branched β-cyclodextrin is recovered from the secondary mother liquor by concentrating or drying the secondary mother liquor to remove the branched β-cyclodextrin. Concentration and drying of the secondary mother liquor to obtain the branched β-cyclodextrin is carried out in a conventional manner using conventional equipment. If a higher purity branched β-cyclodextrin is desired, a compound forming a β-cyclodextrin clathrate may be added to the third solution formed before recovering the branched β-cyclodextrin. is added to form a tertiary precipitate and a tertiary mother liquor. The tertiary mother liquor is then recovered and the branched β-cyclodextrin is recovered from the tertiary mother liquor. The third solution is formed from a secondary mother liquor of between about 1.% and about 20% by weight solids solution, more preferably about 10% by weight solution. Generally, the secondary mother liquor must be concentrated to obtain an approximately 10% solution by weight solids. a third to form a tertiary precipitate and a tertiary mother liquor;
The addition of the β-cyclodextrin clathrate-forming compound to the solution is carried out similarly to the addition of the β-cyclodextrin clathrate-forming compound added to the first or starting solution and the second solution. . 100 of β-cyclodextrin in solution
Based on parts by weight, at least about 10% by weight of the compound forming the β-cyclodextrin clathrate
%, and more preferably about 20% by weight, is added to the third solution. The preferred temperature of the third solution during the formation of the tertiary precipitation and tertiary mother liquor is about 10°C.
~about 25°C, and more preferably from about 15°C to about 20°C
It is between. Preferably, the period for forming the tertiary precipitate is about 3 days or more. Toluene is the preferred β-cyclodextrin clathrate-forming compound used in the tertiary precipitation and formation of the tertiary mother liquor. The practical addition of the compound forming the β-cyclodextrin clathrate to the tertiary solution is accomplished in a conventional manner using conventional equipment. Recovery of the tertiary mother liquor from the 3 solutions is carried out using conventional equipment,
This is carried out in a conventional manner, for example using a centrifuge or a filter. Preferably, this is accomplished by concentrating and drying the aqueous solution to reduce it to a dry solid. Such dry products were found to contain about 70% to about 80% branched β-cyclodextrin. Another step in the invention is to perform an extraction step. Such a step is carried out on the primary precipitation before the second addition of the compound forming the β-cyclodextrin clathrate. The extraction step is carried out by adding about 9 parts by weight of water, based on 1 part by weight of precipitate, to the primary precipitate and mixing the water and precipitate at a temperature ranging from about 20<0>C to about 40<0>C. This extraction step is important in that it is carried out without boiling. The mother liquor resulting from this extraction step is then concentrated and dried. Concentrated and dried mother liquor yields branched β-cyclodextrins. The solids or precipitate remaining after the extraction step are further used to form a second solution;
A second addition of a compound forming a β-cyclodextrin clathrate and the remaining processing is then performed. Yet another alternative is to repeat the primary precipitation step before forming the second solution. In this alternative practice, a solution, referred to as the fourth solution for purposes of differentiation from other solutions, is formed by mixing the primary precipitate and water. The solids concentration of this fourth solution is preferably between about 24% and about 40% by weight solids, and more preferably about 30% by weight solids. For this fourth solution, β-cyclodextrin is present in an amount of about 10 parts by weight or more, more preferably about 20 parts by weight, based on 100 parts of β-cyclodextrin in the solution.
A compound forming a cyclodextrin clathrate is added. The preferred β-cyclodextrin clathrate-forming compound used in this fourth addition is p-xylene. Addition is accomplished in a conventional manner using conventional equipment. The temperature of the starting solution is preferably maintained between about 10<0>C and about 25<0>C, more preferably between 15<0>C and 20<0>C. The period for formation of precipitate from this fourth solution ranges from about 1 day to about
It is the 17th. More preferably between about 3 days and 7 days, and most preferably about 3 days or more. For purposes of identification, the precipitate and mother liquor derived from this fourth solution are referred to as quaternary precipitate and quaternary mother liquor. FIG. 1 illustrates the method of the present invention for the formation of a starting solution 8, followed by the addition of a β-cyclodextrin clathrate-forming compound to the starting solution for primary precipitation and formation of a primary mother liquor. The next step is to recover the primary precipitate originating from the starting solution12. A second solution is then formed by heating and stirring the primary precipitate and water 14. The secondary precipitate and secondary mother liquor are
16 formed from the second solution by addition of a compound forming a β-cyclodextrin clathrate to the solution. A secondary mother liquor from the second solution is recovered 18 and, finally, branched β-cyclodextrin is recovered from the secondary mother liquor. FIG. 2 illustrates a preferred embodiment of the invention in which a washing step 22 is performed between the primary precipitate and the formation 14' of a second solution derived from the washed primary precipitate; Steps 16-20 of are performed, and steps 8 and 10 are performed before step 12. FIG. 3 illustrates yet another preferred embodiment of the invention in which a tertiary mother liquor and a tertiary precipitate are formed from a third solution, and the third solution is formed from a secondary mother liquor. A compound forming a β-cyclodextrin clathrate is added to this third solution to form a tertiary precipitate and a tertiary mother liquor. The tertiary precipitate is recovered 26 and the branched β-cyclodextrin is recovered from the tertiary mother liquor. 28. FIG. 4 illustrates the extraction stage. After collecting 12 the primary precipitate from the starting or first solution, the primary precipitate is subjected to step 30. The extraction solution is recovered from the extraction step 32 and subjected to a concentration and drying process to recover the branched β-cyclodextrin 34. The primary precipitate 36 recovered after the extraction step is used to form the second solution 14''. Steps 16 to 20 are performed subsequently after 14''. FIG. 5 illustrates the repetition of the primary precipitation step;
Here, a fourth solution is formed 38 and the mixing of the primary precipitate and water forms a solution having a concentration of between about 24% and 40% by weight, and more preferably about 30% by weight. Addition of a β-cyclodextrin clathrate-forming compound, preferably p-xylene, to the fourth solution forms a quaternary precipitate and a quaternary mother liquor. After the formation of the quaternary precipitate, it is recovered 42 . This quaternary precipitate is then used to create the second solution 1 in Figure 5 according to the procedure described above.
form 4. Example 1 To a starting aqueous solution containing 463 g of carbohydrate as 30% solids, 45 ml of p-xylene was added and stirred for about 20 hours and allowed to stand for an additional 14 days to form a primary precipitate. The solution was then transferred to a Buchner tube under reduced pressure.
Filter on 18.5 cm filter paper in a funnel. The filter cake was washed three times with approximately 100 ml of distilled water each time. The filtrate and washings were discarded and the filter cake was air dried. Approximately 94g air-dried cyclodextrin/
A p-xylene clathrate compound was obtained. The analysis performed on this material is shown in Table 1 below. This analysis can be performed on a conventional HPLC column.

[Table] Kistrin The filter cake was then added to 11 parts (846 ml) of distilled water and stirred with a magnetic rod for about 17 hours to perform the extraction step of the primary precipitate. Undissolved material was removed by filtration and the filter cake was washed three times with 100 ml portions of water. The filter cake was air dried and set aside. The filtrate and washings were combined and evaporated to 10% solids. At this point, approximately
21.1 g of solid was present and with stirring 10
1.21 g of toluene was added to the % solids solution. Stirring was continued for 3 days at room temperature. The precipitate that formed and said precipitate was removed by filtration and washed three times with 24 ml each of water. The precipitate contained approximately 63 g of carbohydrate. The filtrate and washings were boiled to remove toluene, concentrated, and dried at 110°C. This is shown as a filtrate, and the composition of this filtrate is listed in Table 2 below. Precipitates (carbohydrates) from the processing of the previous extraction step
63 g) was dissolved in 567 g (9 parts) of distilled water and boiled to remove the solvent to form a second solution. next,
12.6 g of toluene (carbohydrate solids) while stirring.
20%) was added. After 3 days, the precipitate was removed by filtration, and the resulting filtrate (third
The solution (solution) was kept under 10% toluene of solids for 3 days. The precipitate was removed by filtration, the filtrate and washings were boiled to remove toluene, concentrated and heated to 110°C.
This product was shown as a filtrate. The composition of the filtrate is shown in Table 2 below. Furthermore, the filtrate and the total product recovered from the filtrate are also shown in Table 2 (note that the total value of the filtrate and filtrate does not exactly match the value of the total product). due to measurement errors and statistical deviations).

【table】 [Brief explanation of drawings]

FIG. 1 illustrates the broad aspects of the invention relating to branched β-cyclodextrins, FIG. 2 illustrates the preferred washing steps of the invention, and FIG. FIG. 4 illustrates the additional steps of forming a tertiary precipitate and a tertiary mother liquor according to the invention; FIG.
- illustrates another step according to the invention by which a cyclodextrin can be obtained, and FIG. 5 illustrates yet another further step which can be used in the invention.

Claims (1)

[Scope of Claims] 1. A method for purifying and separating branched β-cyclodextrin, comprising: (a) adding a complexant forming a β-cyclodextrin clathrate to the first solution; forming a primary precipitate and a primary mother liquor; (b) collecting the primary precipitate; (c) mixing water with the primary precipitate to form a second solution; and (d) adding β-cyclodextrin to the second solution. adding a clathrate-forming compound to form a secondary precipitate and a secondary mother liquor; (e) recovering said secondary mother liquor; and (f) collecting a branched β-cyclodextrin from said secondary mother liquor. said method comprising the step of recovering. 2. The method of claim 1, wherein the complex forming the β-cyclodextrin clathrate is selected from the group consisting of p-xylene and toluene. 3. carrying out step (a) for about 3 days or more;
2. The method of claim 1, wherein the method is carried out at a temperature between about 10<0>C and about 25<0>C. 4. The method of claim 1, wherein both steps (a) and (d) are carried out at a temperature between about 10<0>C and about 25<0>C for about 3 days or more. 5. The amount of β-cyclodextrin clathrate-forming compound added in both steps (a) and (b) is approximately 2. The method of claim 1, wherein the amount is 20 parts by weight. 6. The method of claim 1, further comprising the step of washing said primary precipitate after step (b) and before step (c). 7. A method for purifying and separating branched β-cyclodextrin, which comprises: (a) adding a compound (complexant) forming a β-cyclodextrin clathrate to the first solution to form a primary precipitate and a primary mother liquor; (b) collecting the primary precipitate; (c) mixing water with the primary precipitate to form a second solution; and (d) forming a β-cyclodextrin clathrate in the second solution. adding a compound to form a secondary precipitate and a secondary mother liquor; (e) recovering said secondary mother liquor; and (f) forming a third solution from said secondary mother liquor; (g) adding to the third solution. adding a compound that forms a β-cyclodextrin clathrate to form a tertiary precipitate and a tertiary mother liquor; (h) recovering said tertiary mother liquor; and (i) a β-cyclodextrin branched from said tertiary mother liquor; said method comprising the step of recovering. 8. The method of claim 7, wherein the complex forming the β-cyclodextrin clathrate is selected from the group consisting of p-xylene and toluene. 9. The addition of the β-cyclodextrin clathrate-forming complex in steps (a), (b) and (h) is carried out for about 3 days at about 10°C to about
8. A method according to claim 7, carried out at a temperature of between 25[deg.]C. 10 The amount of the compound forming the β-cyclodextrin clathrate compound added in steps (a), (b) and (h) is about 20 parts by weight based on 100 parts by weight of β-cyclodextrin present in the solution. Claim 7
Method described.